We propose to continue our studies aimed at elucidating the mechanisms by which the E. coli DnaK, DnaJ and GrpE heat shock proteins function as molecular chaperones in various protein metabolic processes. These chaperonins have been highly conserved during 3 billion years of evolution and are known to play vital roles in cells of all living organisms. It is also known that these proteins function both in normal cellular physiology and in response to cellular stress, yet the mechanisms by which they act are understood only in a fragmentary manner. DnaK is the hsp7O homologue of E. coli. It has a weak intrinsic ATPase activity that is stimulated by peptides and by the DnaJ and GrpE co- chaperonins. We propose to continue structure-function studies of the C- terminal peptide-binding domain of DnaK. We will select mutations in this domain that affect interaction of DnaKC with substrate proteins and subsequently assess the functional properties of full-length DnaK proteins harboring such alterations. We will isolate and characterize mutant DnaJ proteins. Two different regions of DnaJ will be targeted for localized mutagenesis. The first region consists of a flexible 30-amino-acid segment rich in glycine and phenylalanine. This segment connects two protease-resistant domains of DnaJ and is essential to DnaJ's capacity to activate DnaK's ATPase. We will also localize the region of DnaJ required to bind to physiological protein substrates of the DnaK/DnaJ/GrpE chaperone system and carry out mutagenesis of this region. Mutant proteins will be characterized for their capacities to interact with DnaK and to bind to specific substrates such as the phage X preinitiation replication complex. We will continue our kinetic analysis of the intrinsic ATPase of DnaK and explore the coupling of the ATPase cycle to the binding and release of polypeptide substrates. Stopped-flow spectrofluorimetry will be used to monitor the kinetics of binding and release of ATP, ADP and peptides to DnaK in the presence and absence of the DnaJ and/or GrpE co-chaperonins. The binding to and dissociation from DnaK of the DnaJ and GrpE chaperones as a function of the DnaK ATPase cycle will also be examined. The possibility that DnaJ stimulates the self-assembly of DnaK into an activated oligomeric form will be explored.